ABSTRACT Histone chaperones escort histones into and out of chromatin, the physiological form of the eukaryotic genome. Many chaperones use acidic intrinsically disordered regions (IDRs) to interact with histones. These IDRs contain the post-translational modification glutamylation. Glutamylation?s role in modulating chaperone function is poorly understood. The perplexing observations that chaperone mono-glutamylation?catalyzed by TTLL4 and removed by CCP5?enhances both histone affinity and deposition highlight a central enigma of histone chaperone mechanisms: the tighter that a chaperone binds to histones, the better a chaperone is at releasing them into chromatin. To explain this, our studies suggest that histone chaperones use their glutamylated acidic IDRs as a DNA-like surface to: 1) capture histones; 2) shield non-specific histone interactions; and 3) structurally stabilize and orient bound histones. Therefore, we hypothesize that glutamylated chaperone IDRs mimic DNA to facilitate binding and pre-organization of histones into a nucleosome-compatible conformation. We will test this hypothesis in the following aims. In Aim 1, we will determine how glutamylated acidic IDRs from the chaperones Nap1 and Nucleoplasmin (Npm2) modulate histone H2A/H2B and linker histone interactions. Using our comprehensive in vitro platform for studying the disorder and function of histone chaperones, we will determine how chaperone IDR glutamylation modulates its histone interaction affinity; we will use NMR to determine where glutamylated IDRs interact with histones and how IDRs structurally stabilize histones; and we will measure the impact of chaperone glutamylation on chromatin assembly in vitro. In Aim 2, we will probe the biological function of chaperone acidic IDR glutamylation. Using our Xenopus laevis cell-free extract model, we will determine when and how chaperone glutamylation modulates chromatin assembly in vivo. We will: identify sites of endogenous chaperone glutamylation; probe the regulation and biological role of chaperone glutamylation during the cell cycle; and measure the impact of chaperone glutamylation on chromatin assembly in egg extract. Our study will move the field forward by answering the central enigma of chaperone function: how is it that the best chaperones for depositing histones also bind histones the tightest? We will similarly advance our knowledge by understanding a novel means of regulating histone chaperone function. Because histone chaperones are critical for genome integrity and gene expression, our work has wide significance. Understanding how chaperones are regulated during the cell cycle will provide key insight for studies of animal development, chromosomal replication, gene expression, and damage repair. IDRs and their PTMs?found in over 30% of eukaryotic proteins?are key components of many regulatory processes, such as liquid-liquid phase-separations. Chaperones are now an important example of this class of proteins. As our work will contribute to the emerging and generalizable concept that the dynamic nature of disordered protein regions is at the core of their function, this proposal is broadly significant.